Abstract
Methanol tolerance of lipase is one of the important factors affecting its esterification ability in biodiesel preparation. By B factor indicated prediction of Candida antarctica lipase B (CalB) surface amino acids, eight sites (Val139, Ala146, Leu147, Pro218, Val286, Ala287, Val306, and Gly307) with high B value indicating more flexibility were chosen to perform saturation mutagenesis. High-methanol-tolerant variants, CalB-P218W and -V306N, created larger haloes on emulsified tributyrin solid plate including 15% (v/v) methanol and showed 19% and 31% higher activity over wild-type CalB (CalB-WT), respectively. By modeling, a newly formed hydrogen bond in CalB-V306N and hydrophobic force in CalB-P218W contributing more stability in protein may have resulted in increased methanol tolerance. CalB-P218W and -V306N transesterified the soybean oil into biodiesel at 30°C by 85% and 89% yield, respectively, over 82% by CalB-WT for 24h reactions. These results may provide a basis for molecular engineering of CalB and expand its applications in fuel industries. The as-developed semi-rational method could be utilized to enhance the stabilities of many other industrial enzymes.
Highlights
Biodiesel produced through transesterification of fats, vegetable or microalgae oils has received increasing interest in the last two decades as a renewable, biodegradable, and non-toxic fuel [1]
A newly formed hydrogen bond in Candida antarctica lipase B (CalB)-V306N and hydrophobic force in CalB-P218W contributing more stability in protein may have resulted in increased methanol tolerance
CalB-P218W and -V306N transesterified the soybean oil into biodiesel at 30 °C by 85% and 89% yield, respectively, over 82% by CalB-WT for 24 h reactions
Summary
Biodiesel (fatty acid methyl esters, FAMEs) produced through transesterification of fats, vegetable or microalgae oils has received increasing interest in the last two decades as a renewable, biodegradable, and non-toxic fuel [1]. The reaction mixture for transesterification is hydrophobic and has a high concentration of methanol or ethanol to get a high rate of conversion of oil to FAMEs. The reaction mixture for transesterification is hydrophobic and has a high concentration of methanol or ethanol to get a high rate of conversion of oil to FAMEs In such an environment, the biocatalyst’s structure tends to denature [6]. The biocatalyst’s structure tends to denature [6] For this reason, searching for high methanol tolerant lipases from the natural world [7, 8], or improving the existing lower-methanol-tolerant lipases into hyper-ones has become meaningful to achieve a competitive biodiesel and cost-effective production technology [9]
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